RNA-sequencing

NIH 3T12 fibroblasts were infected with RDV-50.stop or WT MHV68 at MOI 3 for 18 hours. Samples were harvested in Trizol, chloroform-extracted, and then RNA was isolated with the Qiagen RNeasy kit including the on-column DNase I treatment. Library preparation and sequencing were conducted at Azenta Life Sciences (South Plainfield, NJ, USA).

Extracted RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA).

RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented at 94 °C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).

RNA-seq data were aligned and counted using the CCR Collaborative Bioinformatics Resource (CCBR) in-house pipeline (https://github.com/CCBR/Pipeliner). Briefly, reads were trimmed of low-quality bases and adapter sequences were removed using Cutadapt v1.18 (http://gensoft.pasteur.fr/docs/cutadapt/1.18). Mapping of reads to custom reference hybrid genome described below was performed using STAR v2.7.0f in 2-pass mode^70,71. Then, RSEM v1.3.0 was used to quantify gene-level expression ⁷² with quantile normalization and differential expression of genes analysis performed using limma-voom v3.38.3⁷³. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession GSExxxxx.

The custom reference genome allowing quantification of both viral and host expression used in this alignment consisted of the mouse reference genome (mm10/Apr. 2019/GRCm38) FASTA with a MHV68 FASTA sequence added as an additional pseudochromosome (Supplementary File “MHV68_Krug.fa”). This viral genome was prepared from the annotated herpesvirus genome (NCBI reference) with the addition of a CMV-driven Histone H2B-YFP fusion protein locus found in our mutant MHV68 virus strain, utilized to track individually infected cells. The custom gene annotations used for gene expression quantification consisted of a concatenation of the mm10 GENCODE annotation version M21⁷⁴ and annotations of the MHV68 genome. All overlapping regions of the viral ORFs were removed to create a minimal, non-overlapping annotation. The sequencing library used was unstranded, making stranded counts of highly-overlapping viral ORFs problematic. This annotation was therefore used to make conservative estimates of the expression of individual viral genes. The custom viral GTF annotation files used for this quantification is provided as Supplemental File “M21_MHV68_Krug_Nonoverlapping.gtf”.

Data visualizations for RNAseq were created using Prism (GraphPad), and R (https://www.R-project.org/)⁷⁵, RStudio (http://www.rstudio.com/)⁷⁶, and the pheatmap package (https://CRAN.R-project.org/package=pheatmap)⁷⁷. For both visualizations, counts were normalized first for library size, counts per million mapped reads (CPM), then for composition bias, trimmed-mean of M-values (TMM) before import to RStudio where they were log₂ transformed prior to heatmap creation using the pheatmap package. For the purposes of visualization, +1 was added to each CPM TMM value prior to log₂ transformation.